PROJECT SUMMARY: B cells as a major source of IL-27 cytokine in antibody responses and anti-viral immunity This proposal aims to understand a new regulatory role of B lymphocytes, i.e., induction of these cells to produce IL-27 cytokine, underlying molecular mechanisms, and function of B cell-produced IL-27 in the antibody response and antiviral immunity. As critical immune effectors, B cells integrate innate and adaptive receptor signals triggered by infections to undergo differentiation for antibody responses, such as class-switching to generate different IgG isotypes. These include IgG2a/IgG2c in the mouse, an IgG isotype with potent anti-viral activities and implicated in pathogenic autoantibody response. B cells are also important regulators through production of cytokines that orchestrate immune responses, such as IL-10, IL-35 and ? as we contend here ? IL-27. This cytokine (a heterodimer of IL27p28 and EBI3) functions through its receptor (composed of IL27Ra and gp130) expressed on multiple cell types, including B and T cells. While roles of IL-27 on many T cell subsets have been comprehensively studied, the impact of IL-27 on B cell differentiation only begins to be understood. As indicated by others? findings and our preliminary data shown here, IL-27 induces T-bet transcription factor and IgG2a class-switching in B cells in vitro as well as plays an important role in anti-viral IgG2a responses in vivo. The extensive functional studies of IL-27 contrast the paucity of data on the cellular sources of this cytokine. IL-27 has been considered produced mainly by myeloid cells. However, here we hypothesize that B cells are a previously unrecognized, and likely major, source of IL-27. The rationale stems from our compelling preliminary data showing that in vivo Il27p28 and Ebi3 gene expression as well as secretion of the IL27p28/EBI3 heterodimer depended on B cells. Also, B cells accounted for the majority of IL-27p28 and EBI3 intracellular staining signals. Further, purified human and mouse B cells could be induced to express IL-27 genes and secret IL-27 upon co- stimulation by a TLR ligand and CD154 (CD40 ligand) in vitro. Finally, the synergistic IL-27 induction by TLR/CD40 was augment by IL-21, a hallmark cytokine of T follicular helper (Tfh) cells, in both human and mouse B cells, suggesting an evolutionarily conserved role of IL-21 in a new modality of B cell integrating innate and adaptive signals, namely B cells are primed by TLRs first and then engaged by Tfh cells (Fig. 1). To test our hypothesis, we will address induction of B cell IL-27 production by sequential stimulation, first with a TLR ligand and then CD154 and IL-21, and model contacts of TLR-primed B cells and Tfh cells using a supported lipid bilayer system (Aim 1.1). We will also explore mechanisms underlying the TLR/CD40 synergy, with focus on the role of the RIP3 kinase in NF-kB activation (Aim 1.2). Further, we will analyze and characterize B cells that produce IL-27 at high levels upon immunization or vaccinia virus infection in vivo using Il27p28+/Gfp reporter mice and single-cell transcript analysis (Aim 2.1). Finally, we will address the functional relevance of B cell production of IL-27 in IgG2a responses and anti-viral immunity using mice with B cell-specific knockout of Il27p28 or Ebi3 (Aim 2.2). Our findings would advance understanding of new functions of B cells and IL-27.